Ribulose bisphosphate carboxylase is the CO2-fixing enzyme in the Calvin cycle. It contains two types of subunits: the catalytic "large" subunit (Mr=55 kD) and the noncatalytic "small" subunit (Mr=ca 14 kD). The small subunit is synthesized in the cytoplasm as a precursor polypeptide, cleaved upon entry into the chloroplast, and assembled into holoenzyme. The large subunit is synthesized in the chloroplast, where it binds to a heterologous protein complex consisting of about 12 60 kD polypeptide subunits. This complex sediments at 29S in sucrose gradients and is well separated from the 18S carboxylase and from the 7S free large subunit pool and 3S small subunit pool. We propose to determine the mechanism of assembly of the enzyme by exploiting an in vitro assembly system we have developed from chloroplast extracts. In this system, large subunits can be released from the 29S complex by incubation with ATP, and their assembly into holoenzyme can be facilitated by nucleotides. We will fractionate the extract to separate the 29S complex from the free 7S large subunits and 3S small subunits to see if assembly can occur in the absence of the 29S complex. We will obtain de novo synthesized large subunits by in vitro transcription-translation of cloned large subunit genes, and add these to chloroplast extracts to see if they can assemble into holoenzyme either in the presence of the 29S complex or in its absence. We will also purify the 29S complex in bulk (it is an abundent protein) and study its association-dissociation behavior in vitro, both in isolation and together with either native or holoenzyme-derived large and small subunits. This may lead to the first in vitro re-assembly of this enzyme. The role of heterologous proteins in the assembly of eukaryotic oliogomeric proteins is a relatively unknown field, and is of general significance in cell and molecular biology. This carboxylase assembly system may contribute a lot to our understanding of this general phenomenon, because the components are soluble and well defined.